Project Abstract The central goal is to determine whether glutamate signaling is disrupted in different striatal circuits that underlie repetitive behaviors in mouse models of autism. Biosensor technology will be employed concomitantly with behavioral testing to determine real-time glutamate changes in the striatum during learning, reversal learning and marble burying. Restricted and repetitive behaviors are common to autism spectrum disorders (ASD) but have considerable heterogeneity that can vary in severity and type. A varying severity of cognitive impairment may arise from the degree of heightened dependence on positive reinforcement and increased salience to unpredicted non-reinforcement. This can lead to either a learning deficit or inflexible behavior. Developing probabilistic learning tests for mouse models that match those used to test ASD individuals, we have captured some of the cognitive heterogeneity reported in ASD by testing SHANK3+/- and BTBR mice. SHANK3+/- mice exhibit a probabilistic learning deficit while BTBR mice exhibit a selective probabilistic reversal learning deficit. In a complementary way, we found that BTBR and SHANK3+/- mice exhibit elevated marble burying behavior, but BTBR mice have greater levels than SHANK3+/- mice. To date, there are significant gaps in our knowledge of what neural circuitry and neurochemical mechanisms are altered that underlie repetitive behaviors in ASD. Accumulating evidence indicates that abnormal striatal circuits may underlie certain repetitive behaviors. Further, a long-standing hypothesis to explain ASD features, including cognitive deficits, is an imbalance in the brain excitation/inhibition ratio. There are different lines of evidence that support this hypothesis, although at present, there have been no direct real-time glutamate measurements during behavioral expression of the symptoms. The proposed project will for the first time in two different mouse models of ASD directly examine dynamic changes in striatal glutamate signaling during cognitive tests and expression of a stereotyped behavior. Specific Aim 1 will determine whether real-time glutamate signaling in the dorsomedial striatum, dorsolateral striatum or nucleus accumbens of SHANK3+/- and BTBR mice is altered during spatial learning and reversal learning under conditions in which feedback is certain (100% accurate) and feedback is uncertain (80% accurate for correct choice). Specific Aim 2 will determine in SHANK3+/- and BTBR mice whether glutamate signaling differs in striatal subregions during marble burying behavior. Overall, examination of in vivo glutamate transmission during behavioral testing can provide a better mechanistic understanding of ASD pathophysiology and identify novel therapeutic targets in a disorder known to have heterogeneous symptomology.